Yu-Cheng Ho

Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)-induced disinhibition

Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX2 receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30–300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX2 [N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (compound 29)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a cannabinoid 1 (CB1) receptor agonist. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM 251), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1′-biphenyl]-3-ylcarbamate (URB602), which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). Moderate DAGLα, but not DAGLβ, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM 251. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of 2-AG, an endocannabinoid, through a Gq-protein-mediated PLC–DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.

Orexins/Hypocretins: Pain Regulation and Cellular Actions

Orexin A and B (also named hypocretin 1 and 2) are 33 and 28 amino acid-containing neuropeptides, respectively, derived from prepro-orexin (prepro-hypocretin) which is localized in the the lateral and perifonical areas of the hypothalamus. Two G-protein coupled receptor subtypes, OX1 and OX2, were identified. Orexin-containing fibers and OX receptors are widely distributed in the central nervous system. Orexins have been implicated in the arousal, rewarding, energy homeostasis, autonomic central control and antinociceptive systems. Subtype-selective peptide agonists and antagonists and non-peptide antagonists, but not non-peptide agonists, have been developed. This review summarizes the studies investigating the antinociceptive effects of orexins in various animal models of pain, including trigeminovascular pain, and their cellular mechanisms. Orexins are antinociceptive at both spinal and supraspinal levels. The antinociceptive effect of orexin A is comparable to opioids but orexin B is less or not effective. This effect is opioid-independent and mainly mediated through OX1 receptors. Some animal studies suggest that endogenous orexins may be released during postoperative and inflammatory, but not acute, pain states, or during some stress conditions, which may contribute to stress-induced analgesia. Purinergic P(2X) and glycine receptors are proposed to be involved in orexin-induced spinal antinociception. The supraspinal sites of actions might involve the posterior hypothalamus, which contributes to the trigeminovascular nociception, and the ventrolateral periaqueductal gray, which mediates descending pain inhibition. Endocannobinoids and nociceptin/orphanin FQ were found to interplay with orexins in nocicpetive processing. Further studies are required to elucidate the receptor subtype-specific mechanism(s) and clinical implications of orexin-induced antinociception.

Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition

Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro‐nociceptive in the periphery but is anti‐nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti‐nociception.

Hypofunction of Glutamatergic Neurotransmission in the Periaqueductal Gray Contributes to Nerve-Injury-Induced Neuropathic Pain

Neuropathic pain, a chronic pain due to neuronal lesion, remains unaltered even after the injury-induced spinal afferent discharges have declined, suggesting an involvement of supraspinal dysfunction. The midbrain ventrolateral periaqueductal gray (vlPAG) is known to be a crucial supraspinal region for initiating descending pain inhibition, but its role in neuropathic pain remains unclear. Therefore, here we examined neuroplastic changes in the vlPAG of midbrain slices isolated from neuropathic rats induced by L5/L6 spinal nerve ligation (SNL) via electrophysiological and neurochemical approaches. Significant mechanical hypersensitivity was induced in rats 2 d after SNL and lasted for >14 d. Compared with the sham-operated group, vlPAG slices from neuropathic rats 3 and 10 days after SNL displayed smaller EPSCs with prolonged latency, less frequent and smaller miniature EPSCs, higher paired-pulse ratio of EPSCs, smaller AMPAR-mediated EPSCs, smaller AMPA currents, greater NMDAR-mediated EPSCs, greater NMDA currents, lower AMPAR-mediated/NMDAR-mediated ratios, and upregulation of the NR1 and NR2B subunits, but not the NR2A, GluR1, or GluR2 subunits, of glutamate receptors. There were no significant differences between day 3 and day 10 neuropathic groups. These results suggest that SNL leads to hypoglutamatergic neurotransmission in the vlPAG resulting from both presynaptic and postsynaptic mechanisms. Upregulation of NMDARs might contribute to hypofunction of AMPARs via subcellular redistribution. Long-term hypoglutamatergic function in the vlPAG may lead to persistent reduction of descending pain inhibition, resulting in chronic neuropathic pain.

Targeting the cannabinoid system for pain relief

Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB1) receptors, as well as peripheral CB1 and CB2 receptors. CB2-selective agonists have the benefit of lacking CB1 receptor-mediated CNS side effects. Anandamide and 2-arachidonoylglycerol (2-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, we also review recent studies that revealed a novel analgesic mechanism, involving 2-AG in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. This GqPCR-PLC-DAGL-2-AG retrograde disinhibition mechanism in the PAG can be initiated by activating type 5 metabotropic glutamate receptor (mGluR5), muscarinic acetylcholine (M1/M3), and orexin (OX1) receptors. mGluR5-mediated disinhibition can be initiated by glutamate transporter inhibitors, or indirectly by substance P, neurotensin, cholecystokinin, capsaicin, and AM404, the bioactive metabolite of acetaminophen in the brain. The putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed.

No more pain upon Gq-protein-coupled receptor activation: role of endocannabinoids.

Marijuana has been used to relieve pain for centuries. The analgesic mechanism of its constituents, the cannabinoids, was only revealed after the discovery of cannabinoid receptors (CB1 and CB2) two decades ago. The subsequent identification of the endocannabinoids, anandamide and 2‐arachidonoylglycerol (2‐AG), and their biosynthetic and degradation enzymes discloses the therapeutic potential of compounds targeting the endocannabinoid system for pain control. Inhibitors of the anandamide and 2‐AG degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, respectively, may be superior to direct cannabinoid receptor ligands as endocannabinoids are synthesized on demand and rapidly degraded, focusing action at generating sites. Recently, a promising strategy for pain relief was revealed in the periaqueductal gray (PAG). It is initiated by Gq‐protein‐coupled receptor (GqPCR) activation of the phospholipase C–diacylglycerol lipase enzymatic cascade, generating 2‐AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. Here, we introduce the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, particularly in the PAG. We also review recent studies disclosing the GqPCR–phospholipase C–diacylglycerol lipase–2‐AG retrograde disinhibition mechanism in the PAG, induced by activating several GqPCRs, including metabotropic glutamatergic (type 5 metabotropic glutamate receptor), muscarinic acetylcholine (M1/M3), and orexin 1 receptors. Disinhibition mediated by type 5 metabotropic glutamate receptor can be initiated by glutamate transporter inhibitors or indirectly by substance P, neurotensin, cholecystokinin and capsaicin. Finally, the putative role of 2‐AG generated after activating the above neurotransmitter receptors in stress‐induced analgesia is discussed.

Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation

Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury. SIGNIFICANCE STATEMENT Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.

Tet1-dependent epigenetic modification of BDNF expression in dorsal horn neurons mediates neuropathic pain in rats.

Ten-eleven translocation methylcytosine dioxygenase 1 (Tet1) mediates the conversion of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC), hence promoting DNA demethylation. Although recent studies have linked the DNA demethylation of specific genes to pain hypersensitivity, the role of spinal Tet1-dependent DNA demethylation in nociception hypersensitivity development remains elusive. Here, we report correlated with behavioral allodynia, spinal nerve ligation (SNL) upregulated Tet1 expression in dorsal horn neurons that hydroxylate 5 mC to 5 hmC at CpG dinucleotides in the bdnf promoter to promote spinal BDNF expression at day 7 after operation. Focal knockdown of spinal Tet1 expression decreased Tet1 binding and 5 hmC enrichment, further increased 5 mC enrichment at CpG sites in the bdnf promoter and decreased spinal BDNF expression accompanied by the alleviation of the developed allodynia. Moreover, at day 7 after operation, SNL-enhanced Tet1 expression also inhibited the binding of DNA methyltransferases (DNMTs, i.e., DNMT1, DNMT3a, and DNMT3b) to the bdnf promoter, a requirement for transcriptional silencing by catalysing 5-cytosine (5C) to 5 mC. Together, these data suggest at CpG sites of the bdnf promoter, SNL-enhanced Tet1 expression promotes DNA demethylation both by converting 5 mC to 5 hmC and inhibiting DNMT binding to regulate spinal BDNF expression, hence contributing to behavioral allodynia development.

Growth Arrest and DNA-damage-inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats.

Background: Growth arrest and DNA-damage–inducible protein 45&bgr; reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage–inducible protein 45&bgr;–dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear. Methods: Adult male Sprague–Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage–inducible protein 45&bgr; messenger RNA–targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage–inducible protein 45&bgr;, Ro 25–6981 (an NR2B-bearing N-methyl-D-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings. Results: Nerve ligation-enhanced growth arrest and DNA-damage–inducible protein 45&bgr; expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage–inducible protein 45&bgr; expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage–inducible protein 45&bgr; to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-D-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage–inducible protein 45&bgr; expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25–6981, KN-93, or a growth arrest and DNA-damage–inducible protein 45&bgr;–targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels. Conclusions: By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-D-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage–inducible protein 45&bgr; pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.

Impairment of adenylyl cyclase-mediated glutamatergic synaptic plasticity in the periaqueductal grey in a rat model of neuropathic pain

Long‐lasting neuropathic pain has been attributed to elevated neuronal plasticity changes in spinal, peripheral and cortical levels. Here, we found that reduced neuronal plasticity in the ventrolateral periaqueductal grey (vlPAG), a midbrain region important for initiating descending pain inhibition, may also contribute to neuropathic pain. Forskolin‐ and isoproterenol (isoprenaline)‐elicited EPSC potentiation was impaired in the vlPAG of a rat model of neuropathic pain induced by spinal nerve injury. Down‐regulation of adenylyl cyclase–cAMP– PKA signalling, due to impaired adenylyl cyclase, but not phosphodiesterase, in glutamatergic terminals may contribute to the hypofunction of excitatory synaptic plasticity in the vlPAG of neuropathic rats and the subsequent descending pain inhibition, ultimately leading to long‐lasting neuropathic pain. Our results suggest that drugs that activate adenylyl cyclase in the vlPAG have the potential for relieving neuropathic pain.